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Constraining the Grim Reaper – targeted proteolysis restricts hypersensitive cell death

Principal Supervisor: Professor Murray Grant - SLS

Co-supervisors: Alex Jones - SLS

PhD project title: Constraining the Grim Reaper – targeted proteolysis restricts hypersensitive cell death

University of Registration: Warwick

Project outline:

Classical plant nucleotide binding leucine rich repeat disease resistance genes (NLRs) underpin global food security. They are deployed by breeders to protect elite crops but are rapidly broken down in the field. This project addresses a fundamental challenge in molecular plant pathology; how activated NLRs function and are regulated. A detailed understanding of NLR regulation will complement conventional breeding strategies through targeted engineering of crop disease resistance. This proposal will biochemically and structurally characterise two novel RPM1 (Resistance to Pseudomonas syringae pv. maculicola 1) interacting proteins RIN12 and RIN13. They are predicted to play key and complementary roles in proteolytic control of RPM1 signalling. This is a unique opportunity to study, for the first time, the role for targeted proteolysis in regulation of NLR signaling.

Overview: RIN12 and RIN13 were identified in a screen using the central RPM1 NB-ARC domain - a domain exposed following elicitor induced conformational changes of RPM1. Extensive preliminary data, some of which is summarised below, suggest RIN12 has a gatekeeper in maintaining RPM1 in an inactive configuration and RIN13 acts to specifically degrade activated RPM1 signalling complexes.

RIN12 is a small protease inhibitor of the potato PIN1 class. Over-expression of RIN12 shows a delayed HR and enhanced virulence following DC3000avrRpm1 challenge. Conversely, RIN12 antisense results in a faster HR. Unlike classical PIN1 type inhibitors RIN12 encodes a proline at the key active site “combining loop” and surprisingly unlike PIN1, RIN12 inhibits subtilisin (Fig. 1A) but not trypsin or chymotrypsin activity. Mutating key active site residues results in loss of subtilisin inhibitory activity and ability binding to RPM1 in yeast. Thus we predict that;

  • the RIN12-RPM1 interaction is mediated through the “unique” combining loop of RIN12 and RIN12ox lines sequester all RPM1 in an inactive conformation whereas RIN12as lines have enhanced RPM1 activity or (ii) a lowered threshold for activation of an unknown protease.

PIC1

Our data support a model in which RIN12 prevents inappropriate activation of RPM1, i.e. functioning as a “gatekeeper”. This project addresses 3 specific RIN12 objectives; a) To demonstrate that RIN12 subtilisin-like activity is necessary and sufficient for normal RPM1 function. b) To determine the structures of the RIN12 and RIN12 variants and model the effect of the mutations. c) To attempt the first crystal structure of this small NLR interactor bound to the NB-ARC domain.

RIN13, binds RPM1 via its C-terminus. Remarkably, prokaryotic expressed N-terminal, but not C-terminal, tagged RIN13 is self-cleaved, implicating the C-terminus in autocatalysis and suggesting RIN13 tethered to RPM1 would be inactive. Supernatant from RIN13ox but not Col-5 specifically degrades full length RPM1-MYC; fragments of RPM1-HA but not control proteins (caltractin or Pto kinase) in crude yeast extracts and AvrRpm1-HA but not RIN4 expressed in yeast (Fig. 1B). Thus RIN13 itself, or a protease(s) activated by RIN13, can target specific, sequence unrelated components of the RPM1 signalling complex. We predict RIN13 is tethered to RPM1 and is released via a conformational change leading to autocatalytic cleavage and degradation of RPM1. Thus RIN13 functions in a postactivation desensitization through a tightly controlled negative feedback loop to degrade RPM1 and prevent uncontrolled cell death.

We will test this by:

(i) Characterising the dynamics of RIN13ox SN mediated degradation of RPM1 in Nicotiana benthamiana using YFP labelled RPM1 derivatives.

(ii) Characterising the nature of the protease activity inherent in RIN13ox supernatants

(iii) Fractionating and characterising RPM1 processing activity from RIN13ox supernatant.

(iv) Mapping RIN13 cleavage sites to identify the RIN13 propeptide.

Collectively, these experiments will significantly advance our knowledge of activated NLR function.

BBSRC Strategic Research Priority: Food security

Techniques that will be undertaken during the project:

Protein expression, 2-hybrid interactions, peptide mapping, crystallisation, transient expression in Nicotiana, generation of stable transformed Arabidopsis, biochemistry (protease and protease inhibitor assays, chromatography; affinity & size exclusion), mass spectrometry.

Contact: Professor Murray Grant, University of Warwick